Lab News

INCREASING HEMOGLOBIN HBA2 BY REPAIRING THE HBD PROMOTER, PUBLISHED IN ELIFE

Erythrocytes, or red blood cells, carry hemoglobin and circulate throughout the body to supply oxygen. β-hemoglobinopathies, such as sickle cell disease...

READ MORE

Erythrocytes, or red blood cells, carry hemoglobin and circulate throughout the body to supply oxygen. β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. These conditions result in anemia and organ damage, and available treatment options are limited. Stem cell transplantation is currently the only curative approach, although its feasibility relies on the availability of a suitable donor.

Hemoglobin is a tetrameric protein composed of 2 α-like (HBA) and 2 β-like subunits (HBB). Hemoglobin A1 (HbA1) constitutes 97% of adult hemoglobin, while Hemoglobin A2 (HbA2) makes up 2-3%. HbA2 is composed of two α-globin subunits and two δ-globin (HBD) subunits. HBD is a homologous to HBB gene, but with much lower expression compared to HBB due to a weak promoter.  Currently, many efforts are focused on increasing fetal hemoglobin (HbF) to treat the β-hemoglobinopathies. But HbA2 is more similar to HbA1 and is already expressed at low levels in all adult red blood cells. What if we were to increase HbA2 levels? Could they potentially compensate for beta-globin deficiency? Can genome editing technologies be used to boost transcriptional activity of the endogenous HBD promoter to increase HbA2 levels? Mandy Boontanrart, a Postdoc in our lab, was eager to discover the answers to these questions.

HUDEP-2 cells were edited with CRISPR-Cas9 targeting the HBD promoter to insert transcription factor binding sites. Heterozygous and homozygous clones display increased HBD expression upon insertion of three transcription factor binding sites (KDT).

Using CRISPR-Cas9 genome editing, we inserted various transcription factor binding sequences into the endogenous HBD promoter. Team efforts yielded positive results as we successfully increased the transcriptional activity in HUDEP-2 immortalized erythroid progenitor cells, resulting in a significant upregulation of HBD expression. Despite roughly equal homology-directed repair rates between all promoter designs, we observed a significant increase in HBD only for the design with all three elements (KLF1, β-DRF, and TFIIB). We next explored whether endogenous editing of the HBD promoter can be accomplished in bone marrow stem cells. We found up to 46% HBD expression in clonal populations. We also tested a small molecule drug that enhances HDR outcomes by inhibiting the NHEJ pathway and observed an increase in the percent of HDR alleles in pooled edited bone marrow stem cells.

While our findings provide key mechanistic insight into the globin gene regulation, several questions remain to be tackled.  Is heterozygous knock-in of the promoter design in β-hemoglobinopathy cells is sufficient to ameliorate disease phenotypes? What is the safety profile of this strategy?

Overall, our work is a promising approach for restoring hemoglobin levels in red blood cells. This strategy might open new therapeutic avenues for to treating beta-hemoglobinopathies in the future.

For more, check out our paper, it is now out in Elife!

Note: Excitingly, Mandy is now leading an ETH spin-off, building upon the findings of the paper, check out their brand-new website https://www.ariyabio.ch/!

X Close

Welcome to Nicola

Nicola received his B.Sc in Biology from ETH Zürich in 2021. He is currently enrolled in the Molecular Health Sciences Master´s degree at ETH Zürich and joined...

READ MORE

Nicola received his B.Sc in Biology from ETH Zürich in 2021. He is currently enrolled in the Molecular Health Sciences Master´s degree at ETH Zürich and joined the Corn lab mid February 2023. For his Master´s thesis, he is working on the design and set-up of a whole-genome CRISPR screen, aimed at understanding the cellular response towards oxidative damage on lncRNA.

X Close

Welcome to Naomi!

Naomi received her Bachelor’s degree in Health Science and Technology from ETH Zurich in 2021. Naomi joined the Corn lab in March 2023 as a Master student. For...

READ MORE

Naomi received her Bachelor’s degree in Health Science and Technology from ETH Zurich in 2021. Naomi joined the Corn lab in March 2023 as a Master student. For her Master’s thesis, she will analyse the influence of different factors, such as chromatin state or cell type, on off-target and DNA repair outcomes, in a high-throughput manner.

 

 

X Close

Welcome to Alessandra!

Alessandra Albertelli received her Master’s degree in Medical Biotechnology and Molecular Medicine from the University of Milan in 2017. In her Master’s...

READ MORE

Alessandra Albertelli received her Master’s degree in Medical Biotechnology and Molecular Medicine from the University of Milan in 2017. In her Master’s thesis, she focused on long intergenic non-coding RNA in lymphocyte T differentiation. She then spent five years at Heidelberg University as a research assistant and gained competence in confocal microscopy, cell differentiation, genome editing, and protein interaction with DNA and RNA.
Alessandra is curious to learn new techniques and is happy to help the lab with her expertise and passion for her job.
Alessandra joined the Corn lab as Research Technician in January 2023.

X Close

Welcome back Martina!

Martina received her MSc in Biology from ETH Zurich in September 2022. She worked in the Corn lab, focusing on the role of the ubiquitin-proteasome system in...

READ MORE

Martina received her MSc in Biology from ETH Zurich in September 2022. She worked in the Corn lab, focusing on the role of the ubiquitin-proteasome system in an autosomal dominant form of cerebral palsy. Martina returned to the Corn lab as a PhD student in December 2022, investigating how chemical modification of the protein backbone affects protein stability. Her research interests include protein homeostasis and functional genomics, as well as the potential application of protein engineering in biotechnology.

X Close

BASE EDITING AS A POTENTIAL CURE FOR FANCONI ANEMIA – PUBLISHED IN NATURE COMMUNICATIONS

Cas-mediated genome editing technology holds great promise as a curative treatment for a number of genetic diseases. Conventional CRISPR-Cas genome editing...

READ MORE

Cas-mediated genome editing technology holds great promise as a curative treatment for a number of genetic diseases. Conventional CRISPR-Cas genome editing induces DNA damage (double stranded DNA breaks) and relies on the cellular DNA repair system to yield the desired repair outcome. This works quite well in cells with a fully operational DNA repair machinery. However, in Fanconi Anemia (FA), a genetic disorder associated with bone marrow failure and cancer predisposition, DNA repair is defective due to the gene mutations causing the disorder. Homology directed repair (HDR)-based editing strategy as an option to correct FA mutations is pretty inefficient (Richardson et al. Nature Genetics 2018). But is it possible to avoid DNA cleavage, and instead use recently developed genome editing systems such as base editing? Can base editing reverse the effects of FA mutations? New work led by a postdoc Erman Karasu (co-corresponding author) and PhD student Sebastian Siegner together with Alexandra Clemens at ETHZ and Laura Ugalde from Paula Rio’s lab shows that this is possible, even in bone marrow stem cells from FA patients.

In this proof-of-concept study, we find that base editing can indeed restore the function of FA bone marrow stem cells. First, the team went through cycles of optimization for the conditions (base editor construct, vector type, guide RNA format, delivery) in cell lines from multiple FA patients. The developed approach effectively corrected FA mutations in both patient-derived cell lines and bone marrow stem cells from FA patients, leading to restored FANCA expression and functional FA pathway and phenotypic resistance to crosslinking agents.

An obvious question that comes up: how safe is this editing approach? To answer this question, the team predicted possible off-targets and measured the editing outcomes in > 60 sites across multiple base editors. Unintended modifications were detected at a single site with one guide RNA, but a guide RNA targeting the most prevalent FA mutation had no detected off-targets.  Nevertheless, un-biased off- target identification using genome or RNA-sequencing will be the next step in the preclinical validation of base editing as an approach to cure FA.

Altogether, this work highlights base editors as a feasible editing strategy in FA and brings us one step closer to the future clinical implementation of base editing not only in FA, but also in other genetic diseases.

Check out our paper, now out in Nature Communications!

X Close

CRISPR-SCREENS IN ORGANELLE AUTOPHAGY- REVIEW PUBLISHED IN TRENDS IN CELL BIOLOGY

Are you interested in autophagy of really big things in the cells, such as organelles?  Are you wondering how cutting-edge genetic tools can accelerate your...

READ MORE

Are you interested in autophagy of really big things in the cells, such as organelles?  Are you wondering how cutting-edge genetic tools can accelerate your autophagy research? Jin Rui Liang (Amos), a former postdoc, now running his own group at the University of Dundee, wrote a comprehensive review on CRISPR’s impact on autophagy research. Amos outlines the major considerations for CRISPR-based genetic manipulations in autophagy, with a focus on genome-wide screening, as well as various reporter systems for high-throughput autophagy quantification. The article thoroughly summarizes all the relevant info on recently performed autophagy-related screens and their discoveries. Have a CRISPR-view on autophagy, now out in Trends in Cell Biology!

X Close

Welcome to David

David received his PhD in Biochemistry from the University of Cambridge in 2022 under the supervision of Prof. Steve Jackson. His work focused on identifying...

READ MORE

David received his PhD in Biochemistry from the University of Cambridge in 2022 under the supervision of Prof. Steve Jackson. His work focused on identifying functional genetic interactions within the DNA damage response through CRISPR-Cas9 screens. David joined the Corn Lab as a postdoctoral researcher in October 2022. 

X Close

Welcome to Jan

Jan received his Master´s degree in Immunology from Charles University in 2022, working with Dr. Petr Kašpárek on the role of TNFR1 signalling in the Netherton...

READ MORE

Jan received his Master´s degree in Immunology from Charles University in 2022, working with Dr. Petr Kašpárek on the role of TNFR1 signalling in the Netherton syndrome. Jan joined the Corn Lab as a Research Assistant in September 2022 and his research interests include regulation of gene expression and synthetic biology, with a special focus on utilizing the CRISPRa/i technologies in therapeutic applications.

X Close

FILTERS

Tweets

Contact Us

Questions and/or comments about Corn Lab and its activities may be addressed to:

JACOB.CORN@BIOL.ETHZ.CH

Share: